The overall goal of this three-year R33 proposal is to develop a novel proteomic strategy for quantitative comparisons of serum protein profiles capable of detecting proteins down to the low ng/ml level. This is about 100- to 1000-fold more sensitive than direct analysis of serum on either broad range or narrow range 2-D gels, and it represents the lower end of the concentration range for most known tumor serological markers. This high sensitivity proteome strategy will be developed and tested using chimeric mouse serum that contains human proteins secreted by tumors grown subcutaneously from injected human melanoma or pancreatic carcinoma cell lines. Systematic analyses of secreted human proteins using the new methods developed in this project should identify multiple new potential diagnostic targets for cancer screening. This proteome strategy will incorporate several novel supporting methods in addition to further optimization of a very promising proteome fractionation device and method recently invented in our laboratory. The fractionation method, "microscale solution isoelectrofocusing" (microsol-IEF) uses a variable number of tandem small volume chambers separated by semipermeable partitions containing immobilines at specific pH?s. Proof-of-principle experiments have been completed on several types of samples including mouse serum and demonstrate that this method can cleanly fractionate at least several mg of crude extracts into a small number of fractions. The resulting well-resolved fractions can be separated on a series of slightly overlapping narrow range 2-D gels using much higher protein loads than when unfractionated samples are used. Although the feasibility of this prefractionation method has been demonstrated, more reliable devices must be developed and separation parameters must be optimized to maximize resolution and reliability before this method can become a reliable robust proteomics method. Other novel features of the overall proteome analysis strategy include use of custom-made slightly overlapping narrow pH range gradients to ensure proteins are not lost at gel boundaries and use of dual sensitivity stains to increase dynamic range of detected spots. Proteins that appear to be specific to tumor-bearing mice will be identified and their species of origin (mouse = host or human = tumor) will be determined using LC-MS/MS methods. The Specific Aims are: 1) Develop an optimized microsol-IEF device and method, 2) Develop high sample capacity microsol-IEF devices, 3) Develop high sensitivity post microsol-IEF analysis methods, and 4) Systematically apply integrated proteome analysis strategy to serum samples containing human melanoma or pancreatic carcinoma tumors.